Cobalt-assisted route to rare carbocyclic C-ribonucleosides

(Re)emerging RNA viruses have been major threats to public health in the past years, and from the few drugs available, nucleoside analogues are still at the cornerstone of the antiviral therapy. Among them, the synthesis of carbocyclic C-nucleosides is suffering from long syntheses and poor yields. Herein we report a concise stereoselective synthesis of rare carbocyclic C-nucleosides (11a–l) bearing non-canonical nucleobases through a cobalt-assisted-route as key step starting from the optically pure (−)-cyclopentenone 1. This approach paves the route for novel carbocyclic C-nucleoside discovery.


Introduction
The continuous growth of the human population as well as human interaction with wild environments have shown several emerging and re-emerging RNA viruses responsible of highly fatal viral diseases and threatened public health with deadly outbreaks of global concern, [1][2][3][4] the majority of RNA viruses are zoonotic or vector-borne infectious agents [5][6][7] and are considered as the most common class of pathogens (2 to 3 novel viruses discovered by year); 8 they represent a challenge for global disease control 9 because of their faster evolutionary rates and increased mutability compared to some DNA viruses. 10Except for the SARS-CoV-2 for which vaccines have been developed at an unprecedented speed, the biological diversity and rapid adaptive rates of RNA viruses make that no effective commercial vaccine is available for any of the above-mentioned viruses, and we are still awaiting the rst effective antiviral drugs to be approved.Nucleoside analogues, with more than 40 derivatives marketed to treat viral DNA infections as well as certain cancers or bacterial infections, 11 are the most promising and leadingclass of antivirals against RNA emerging viruses. 12On one hand, the replacement of the oxygen in the furanose ring with methylene group led to carbocyclic nucleosides which are chemically more stable towards the enzymatic cleavage of the Nglycosidic bond and have an increased cell permeability because of its higher lipophilicity than their nucleoside parents.The broad spectrum of biological activities of carbocyclic nucleosides and their pharmaceutical importance have generated considerable research interest in their design and synthesis.Many reviews by us 13 and others 14 have been published on this topic, as well as on their biological applications. 15 the other hand, C-nucleosides, 16 in which the ribofuranosyl moiety is linked to a heterocyclic non-canonical base (such as bearing hydrophobic aryl group) 17 through a C-C bond instead of C-N-bond found in natural nucleosides, have been reported for their antiviral and/or anticancer potential.So far, no natural carbocyclic C-nucleosides have been found in nature and only a few have been synthesized to date (Fig. 1), such as the inhibitor of histone methyltransferase DOT1L recently developed by Paruch et al., 18 the carbocyclic formycin 19 analog by Leahy et al., and the compound reported by Fletcher et al. 20 bearing a diuoro pyridine non-canonical nucleobase.
A rst approach was reported in 2001 by Chu and coworkers 21 from 1,4-g-ribonolactone as chiral starting material (Scheme 1A).The key intermediate is obtained by Bu 3 SnH-AIBN reduction of an olenic intermediate obtained by treating the protected ribonolactone with ethyl cyanoacetate and tBuOK and then it is converted to nal carbocyclic C-nucleosides by the full construction of the pyrazole non-canonical nucleobases.
Paruch and co-workers developed a diastereoselective, racemic procedure from norbornadiene through the TIPSprotected cyclopentanone which underwent diastereoselective addition of various lithiated (hetero)aryl moieties (Scheme 1B). 22A recent publication by Fletcher group 20 reported the Rhcatalytic asymmetric synthesis of carbocyclic C-nucleosides using an asymmetric Suzuki-Miyaura type reaction as the key C-C bond forming step, followed by late-stage addition of the hydroxymethyl group to ribose analogs (Scheme 1C).Even if several efforts towards the synthesis of enantioenriched carbocyclic nucleosides have been reported in the past years, the multi-step sequences combined with the difficulty associated to the installation of stereocenters make these approaches long, tedious, and not suitable for the pharmaceutical industry.Thus, the synthetic difficulties have probably hampered the production of those very promising carbocyclic Cnucleoside analogs.
Hence, we focused on designing a new, versatile, robust and stereo-controlled synthetic route to diverse carbocyclic Cnucleosides without the use chiral catalyst but taking advantage of stereocenters previously set in the molecule (Scheme 1D).Several non-canonical aromatic bases were installed. 23This (hetero)aryl moieties included electron-donating and electronwithdrawing substituents, and the presence of heteroatoms in the ring, while maintaining p-stacking and Watson-Crick hydrogen bond interactions.

Results and discussion
We began the synthesis from the commercially available optically pure (−)-cyclopentenone 1 which is easily obtained in 3 steps from D-ribose. 24Following a reported procedure by Schneller et al., 25a the diastereoselective Grignard addition of vinyl magnesium 25b bromide on 1 afforded the ketone 2 as one diastereomer in 63% yield (Scheme 2). 1 H NMR and NOESY experiments revealed that H 3 correlates only with H 2 (d, J(H 3 ,H 2 ) = 5.2 Hz) and not with H 4 corresponding to a dihedral angle close to 90°according to Karplus equation.This angle is attainable with H 3 and H 4 trans to each other (but not when they are cis), proving that the vinyl substituent of the organocuprate added to the less hindered face resulting in 2 with the depicted stereochemistry, as illustrated by molecular models (see ESI †).The use of TBAF in the quenching of the Grignard addition step is vital since the yield of 2 can be negatively affected by the formation and isolation of silyl ether 2 ′ .Reduction of resulting cyclopentanone 2 with AlLiH 4 afforded alcohol 3 in 97% yield.The compound was diastereomerically pure according to 1 H and 13 C NMR. 1 H NMR and NOESY analysis, and Karplus equation indicate that compound 3 has H 1 and H 2 in a cis relationship.25b This is in agreement with molecular models (see ESI †).With alcohol 3 in hand, an Appel-type reaction (proceeding with the inversion of conguration) using iodine and triphenylphosphine in the presence of imidazole was intended.This reaction was initially performed using dichloromethane as solvent, but unfortunately, just starting material was recovered.Finally when toluene was used as solvent and the reaction was done under reux conditions, the desired iodohydrin 4 was obtained in 90% yield.Interestingly, 1 H NMR clearly shows a van der Waals deshielding effect of the iodine atom on the vinylic C-H proton.
Inspired by the cobalt-mediated diastereoselective cross coupling reaction reported by Knochel and co-workers, 26 we decided to apply this methodology as key step of the synthesis of carbocyclic C-nucleosides.According to the authors, the presence of the neighbouring TBS-ether group is responsible for the high diastereoselectivity observed because the silyl ether and the newly inserted aromatic group adopt an anti-conguration.We sought the isopropylidene motive at C2-C3 would have a similar effect by blocking the a-phase of the cyclopentane nucleoside.The cobalt solution can be prepared in a day using the protocol reported by Knochel.Before trying the conditions in the previously synthesized iodohydrin 4, we decided to test them on the model substrate 7 (Scheme 3).Starting from epoxide 5, iodohydrin 6 was obtained in 72% yield.Further protection of the alcohol as a TBS ether afforded the model substrate 7 in 84% yield as a racemate.
With 7 in hand, we tested the cobalt-catalysed cross coupling conditions.Thus, to a cooled solution of the iodohydrin 7 and TMEDA in dry THF was slowly added the Grignard reagent.To our delight, we were able to isolate the desired compound 8 in 88% yield as only relative trans stereochemistry, when the reaction was allowed to stir at room temperature overnight.The aryl group was trans to the silylated ether.We did notice that shorter reaction times resulted in a decreased reaction yield.
We then applied the above-mentioned conditions for the cross-coupling reaction between halohydrin 4 and different Grignard reagents (Scheme 4).The desired products (9a-l) were obtained in good yields in the presence of activating groups, such as methoxide 9c and triuoro-methoxide 9d.Fluorinated compound 9b was isolated in 68% yield.Ortho-substituted product 9e was successfully obtained in moderate yield despite its steric hindrance and the presence of a nitrogenated base.
Binaphthyl substituted products 9f-h were also were also isolated, although in poor to moderate yields.Only one isomer is produced and its formation proceeds through a proposed radical mechanism (Scheme 5). 27ith alkenes 9a-l in hand, an oxidative cleavage of the double bond was attempted.Initially, a ruthenium catalysed reaction was performed on 9a furnishing 10a in just 25% yield.To our delight, the use of osmium tetroxide afforded alcohols 10a-l in excellent yields (Scheme 4).Later deprotection of the isopropylidene ketal in acidic media yielded the desired carbocyclic C-nucleoside analogues 11a-l.It should be noticed that the 1 H and 13 C NMR spectra of 11a are consistent with those reported by Fletcher et al. for the same compound obtained by a different way. 20

Conclusions
In summary, a series of hitherto unknown rare carbocyclic Cnucleosides (11a-l) were successfully synthesized in a highly efficient manner from protected ribonolactone 1 through a cobalt-catalyzed cross-coupling between the enantiopure halohydrine 4 and various aryl Grignard reagents.To the best of our knowledge, the proposed synthetic procedure is far superior to any other route reported to date.It is believed that this study will contribute to explore this promising class of carbocyclic Cnucleosides.

General methods
Commercially available chemicals were of reagent grade and used as received.The reactions were monitored by thin layer chromatography (TLC) analysis using silica gel plates (Kieselgel 60F254, E. Merck).Column chromatography was performed on Silica Gel 60 M (0.040 × 0.063 mm, E. Merck).Optical rotations were measured with a PerkinElmer 341 polarimeter in appropriate solvent, at the indicated temperature (20 °C) and at 589 nm sodium line, in a 1 dm cell.Concentrations are given in g/100 mL.The 1 H and 13 C NMR spectra were recorded on a Varian InovaUnity 400 spectrometer (400 MHz) in (d4) methanol, CDCl 3 , shi values in parts per million relative to SiMe 4 as internal reference.High Resolution Mass spectra were performed on a Bruker maXis mass spectrometer by the "Federation de Recherche" ICOA/CBM (FR2708) platform.

Preparation of the CoCl 2 $2LiCl (1.0 M) solution in THF
The CoCl 2 $2LiCl (1.0 M) solution in THF was prepared following the reported experimental procedure. 24A dried two neck 50 mL ask equipped with a stirring bar a septum was charged with anhydrous LiCl (2.12 g) and heated at 130 °C under high vacuum for 2 h.Aer cooling to room temperature under argon, CoCl 2 (3.24 g) was added.The resulting mixture was heated to 130 °C for 5 h under high vacuum, cooled down to room temperature under argon and charged with dry THF (25 mL).The resulting solution was vigorously stirred at room temperature overnight.The dark blue solution was used within 1 month.

Synthesis of Grignard reagents
All non-commercially available aryl Grignard reagents were prepared following the following experimental procedure.To a ask containing a stirring bar was added Mg turnings (240 mg, 10 mmol, 2.0 equiv.)and LiCl (230 mg, 5.5 mmol, 1.1 equiv.).The ask was ame dried under vacuum and ushed with argon three times.THF (5 mL) and dibromoethane (25 mL) were then added, followed by the dropwise addition of 1-bromo-4-methoxybenzene (935 mg, 5 mmol).The resulting solution was stirred at room temperature for two hours and used immediately in the cross coupling reaction.

General procedure A: cobalt mediated cross coupling reaction
To a stirred solution of iodohydrin (1.0 equiv.) in dry THF (1 M) was added TMEDA (0.3 equiv.)and a 1.0 M solution of CoCl 2 -$2LiCl in THF (0.85 equiv.).The resulting blue solution was cooled down to −78 °C before the Grignard reagent (1.7 equiv.) was slowly added.The reaction mixture was stirred at room temperature overnight.A saturated aqueous solution of NH 4 Cl was added to quench the reaction.The aqueous layer was extracted with AcOEt, and the combined organic extracts were washed with brine, dried over anhydrous MgSO 4 , ltered and concentrated under reduced pressure.The crude mixture was then puried by ash chromatography to afford the desired product.

General procedure B: oxidative cleavage of double bond catalysed by osmium
To a stirred solution of alkene (1.0 equiv.) in a mixture of methanol: water (7 : 3) (0.11 M) was added NaIO 4 (2.07 equiv.).The reaction mixture was cooled down to 0 °C and a 2.5 wt% solution of OsO 4 in t-BuOH (0.08 equiv.) was slowly added.The resulting mixture was stirred 1 h at 0 °C and 2 h at room temperature.The newly formed white solid was removed by ltration and washed with a mixture of methanol water (2 : 1).The ltrate was then cooled down to 0 °C and NaBH 4 (6.4 equiv.) was added portion wise, turning the reaction black.The resulting mixture was allowed to stir at room temperature for 30 minutes, aer which the volatiles were removed under reduced pressure.Brine was then added and the aqueous layer was extracted with CH 2 Cl 2 .The combined organic extracts were washed with brine, dried over anhydrous MgSO 4 , ltered and concentrated under reduced pressure afford the desired product.